New developments in CRISPR technology, with a focus on mouse and human cell applications.

Wednesday, April 23, 2014

#CRISPR guidelines part 2: CRISPR target choice considerations.

The target should typically have the sequence motif N21GG. The first 20 bases form the
“protospacer” and they must to
be incorporated as part of the guide RNA that will localize the Cas9 DNAse to
the target site. These bases are required to pair with the target complementary
DNA strand. The CRISPR/Cas9 mechanism can apparently utilize almost any
sequence for the unspecified bases. Although some target sequences can
apparently exhibit varying cleavage efficiencies, the cleavage efficiency at a
specific site cannot currently be predicted
based on the characteristics of the target sequence. Therefore, there may
be occasions where targets need to be tested empirically.

The last 3 bases of the N21GG
motif (that is, ...NGG) are the PAM (protospacer-
adjacent motif). The last GG dinucleotide is required by the S. pyogenes Cas9 for efficient target
recognition and interacts directly with the Cas9 protein. Deviations from this
motif significantly reduce efficiency of cleavage [8]. In the
near future, Cas9 variants may be engineered that utilize different PAM motifs.
Finally, some Cas9 proteins from other bacterial species can apparently have
different PAM recognition motifs [9], so the array of possible
CRISPR/Cas targets may increase as alternate Cas variants become available.

• Are there required sequence characteristics within
the 20-base base-pairing region? To efficiently synthesize the
guide RNA, the RNA polymerase will usually prefer certain bases in the first 1 or 2 bases of the protospacer
sequence.Note that these are
defined by the type of RNA polymerase used to synthesize the guide RNA, not the
CRISPR/Cas9 mechanism itself.For
in-vitro synthesized guide RNAs, T7 RNA polymerase will be typically
employed.T7 RNA Pol “prefers” GG
as the first two bases.If the guide RNA is to be expressed inside the target cell from an injected
(or transfected) DNA plasmid, the vector will often contain the human U6 snRNA
promoter for this purpose.This
promoter is transcribed by RNA Pol III which prefers G as the first base.

Other than
that, there are apparently few strict limits on the sequence content of the
protospacer.However, efficiency
of guide RNA loading onto Cas9 is optimized when the last 4 protospacer bases
are purines and U is avoided (T in the target sequence) [10].Also, avoid targets with stretches of 4
or more T’s in a row as they can impair guide RNA transcription.TTTTT is the termination signal for RNA
Pol III.

•Can the
protospacer sequence be lengthened to improve efficiency or specificity?No.[11]

• Can the protospacer be shortened?Surprisingly, the protospacer can
apparently be shortened by trimming off 1-3 bases from the 5’ end, with
negligible loss in cleavage efficiency and reduction in off-target cleavage [12].

• Where in the target does Cas9 cleave the DNA?The Cas9-mediated strand cleavage
sites are within the protospacer, close to the PAM motif.Cas9 normally cleaves both
strands.The precise
cleavage site on either strand can vary slightly [2] but map
within the colored regions below:

• Identifying potential CRISPR targets in genes of
interest: The PAM motif is reasonably frequent in mammalian DNA. Many
or most genes will have enough targets to be amenable to some form of
mutagenesis with this motif, so we suggest it as a starting point for target
searching in your gene of interest. Also, a CRISPR target design tool is
available: http://crispr.mit.edu.

In the
near future, S. pyogenes Cas9
variants may be created that utilize different PAM motifs (but not as of early
2014). Finally, some Cas9 proteins from other bacterial species can apparently
have different PAM recognition motifs [9], so the array of possible
CRISPR/Cas targets may increase as alternate Cas variants become available.